The goal of this project is to discover new genes for hearing loss by identifying inherited mutations in multiplex families. Targeted DNA capture and massively parallel sequencing technologies will be used to identify mutations in the deaf population of the Middle East, including 270 Israeli Jews and 212 Palestinian Arabs. We will explore the physiological processes underlying deafness using mouse mutants. This project will be led and directed by Karen Avraham (Tel Aviv) and Moien Kanaan (Bethlehem), who have worked together for 15 years with 11 joint publications on genetic deafness and 2 reviews on peace building through scientific collaboration. Discovery and characterization of new genes for hearing impairment involves five aims. In AIM 1, we will fully sequence 192 affected individuals for 246 genes for syndromic and non-syndromic hearing loss, in humans and mice, with a custom 1.6 MB design of cRNA oligonucleotides. We will use a multiplex barcoding strategy and sequence on an Illumina GAIIx Genome Analyzer. Mutations will be validated by Sanger sequencing. In AIM 2, homozygosity mapping using the Affymetrix 6.0 array will be conducted for families not resolved in Aim 1. In addition to identifying homozygous regions, SNP data will be analyzed to detect homozygous CNVs. In AIM 3, exome sequencing will be carried out and variants that map within homozygous regions, obtained from Aim 2, will be evaluated first. For families with no extended homozygous regions, variants exome-wide will be evaluated for compound heterozygosity. One flow cell lane will be run for each proband. In AIM 4, the targeted pools of Aim 1 will be revised to include new genes for screening of all unresolved probands, familial or sporadic. Clinical features of hearing loss will be compared across mutations and genes. In AIM 5, newly identified proteins will be characterized by expression analysis in lymphoblastoid cell lines, protein-specific functional assays, silencing or overexpression by viral transfection in cochlear cultures;, protein localization throughout development of wildtype mice;hearing and vestibular testing, and inner ear morphology, including high-resolution scanning electron microscopy, in mouse models for human deafness Discovery of new deafness genes will allow for early clinical diagnosis, enabling prediction of phenotypes and enhanced rehabilitation, in particular for children. Characterization of critical proteins will enable a comprehensive understanding of the biological mechanisms involved in pathophysiology of hearing loss. PUBLIC HEALTH RELEVANCE: NARRATIVE The goal of this project is to identify new mutations and genes responsible for inherited hearing loss using state-of-the-art genomic tools such as DNA capture and massively parallel sequencing technologies. Middle Eastern family structure facilitates identification of these genes, which may be present in other populations in the world and thus have far reaching consequences, including in the United States. The research will lead toward enhanced clinical diagnostics, a biological understanding of a comprehensive list of proteins involved in auditory function, and a potential for development of therapeutics for deafness.